The 4-gene score correlated with overall survival of TCGA , which was validated with DFS and disease-specific survival of METABRIC cohort. The 4-gene score was able to predict worse survival or clinically aggressive tumors, such as high Nottingham pathological grade and advanced cancer staging. High score was associated with worse survival in the hormonal receptor (HR)-positive/Her2-negative subtype. High score enriched cell proliferation-related gene sets in GSEA. The score was high in primary tumors that originated, in and metastasized to, brain and lung, and it predicted worse progression-free survival for metastatic tumors. Good tumor response to neoadjuvant chemotherapy or hormonal therapy was accompanied by score reduction. High scores were also predictive of response to neoadjuvant chemotherapy for HR-positive/Her2-negative subtype. High score tumors had increased expression of T cell exhaustion marker genes, suggesting that the score may also be a biomarker for immunotherapy response. Our novel 4-gene score with both prognostic and predictive values may, therefore, be clinically useful particularly in HR-positive breast cancer.We generated a 4-gene score with genes upregulated in LM2-4, a metastatic variant of MDA-MB-231  that was strongly associated with disease-free survival (DFS) in TCGA cohort (hazard ratio [HR]>1.2,  Although breast cancer has a relatively better prognosis compared to other cancers, approximately 40,000 patients die annually from this disease in the US . The mosAlthough treatment for metastatic breast cancer is essential to further improve breast cancer survival, currently there is no cure that can eradicate metastatic disease. On the other hand, treatment for cancer has improved significantly during the last decade, with new regimens and approaches in chemotherapy and immunotherapy bringing considerable benefits for late-stage cancer patients. For example, metastatic melanoma, once considered a fatal diagnosis, has become a curable disease to some with the advent of immunotherapy . Early iThe availability of high-throughput technology that is not economically prohibitive has led to the rapid accumulation of data on transcriptome-wide gene expression of tumors from a large number of patients. Exploration of such data by the research community has been of importance to the identification of potential biomarkers that may be more accurate at the individual patient level . Cancer In this study, we compared the gene expression profiles of poorly metastatic human breast cancer cell line MDA-MB-231) with a highly-metastatic variant (LM2-4) that was selected after multiple rounds of orthotopic implantation into immune compromised mice and then, following surgical removal of a primary tumor, re-selected from spontaneous metastasis in the lung ,23. Tran1 with a The human metastatic breast cell variant LM2-4 selected in vivo from the parental MDA-MB-231 cell line has been extensively described, with studies based on organ tropic seeding to the lungs and how it can be impacted by surgery and treatment detailed elsewhere ,27,28,292 units in LM2-4 compared to MDA-MB-231 with false discovery rate (FDR) < 0.05, whereas the expression was significantly reduced for 250 genes. To understand the relevance of the 297 upregulated genes for metastasis, we examined the association of their expression in treatment-naive primary tumors with disease-free survival (DFS) in the TCGA breast cancer cohort of 1093 patients. DFS was deemed an appropriate indicator of metastasis since the majority of breast cancer recurrences were as metastasis. Using univariate Cox regression analyses we identified four genes, DOK4, HCCS, PGF and SHCBP1, whose tumoral expression was significantly associated with DFS with HR > 1.2 and p < 0.02 (there were 5 genes with p = 0.02–0.05). We generated a multivariate Cox regression model for DFS using expression values of the 4 genes. A 4-gene score using tumor gene expression and Cox regression coefficient values in the model was calculated as: 1.355 × (expressionDOK4) + 1.641 × (expressionHCCS) + 1.345 × (expressionPGF) + 1.232 × (expressionSHCBP1).The expression of 297 genes was higher by ≥ 1.2 logp < 0.001), which was higher than the HR with the individual genes  METABRIC cohort of 1904 breast cancer patients . The valp < 0.001; DSS: HR = 1.57, 95% CI = 1.32–1.85, p < 0.001; To validate the association of the 4-gene score with survival, we applied the scoring model to the METABRIC cohort. We analyzed disease-specific survival (DSS) instead of OS because OS data is not available for METABRIC. Patients with higher 4-gene scores had worse survival, mirroring the result for TCGA cohort  and OS . The independent nature of the 4-gene score was also seen for the METABRIC cohort, for which subtype, stage, grade, and score were significant factors in univariate analyses of DFS and DSS (p = 0.002), and for DSS was 1.39 .We performed univariate and multivariate Cox regression analyses to evaluate if the prognostic value of the 4-gene score is independent of various other clinical and pathological factors. In univariate analysis of DFS in the TCGA cohort, cancer staging (III/IV vs. I/II), and the score (high vs. low) had significant HR . For OS, and DSS . In multp = 0.005, DSS p = 0.015) but also the high-grade group   C. In earAmong the primary breast cancer patients of TCGA, the 4-gene score was higher in TNBC and HER2+ subtypes, which are known to be more clinically aggressive than HR+ and HER2− subtype . This reTo obtain biological insight into the nature of breast cancer that may explain the prognostic and predictive values of the 4-gene score, we conducted gene set enrichment analyses to compare tumors with high and low scores. Tumors with high scores had significant enrichment for 10 of the 50 mSigDb Hallmark gene sets A. The 4 Recently there has been an accumulation of data that the tumor immune microenvironment plays a significant role in cancer biology and behavior ,15,17,20Because our 4-gene score was developed based on the gene expression differences between the MDA-MB-231 and its lung-metastatic variant LM2-4, and because it is prognostic for survival in breast cancer in which distant metastasis is the major cause of death, we hypothesized that the 4-gene score is predictive of the development of distant metastasis. First, we examined the scores in a cohort of primary breast cancer tumors that eventually developed distant metastasis to bone alone, brain, or lung (GSE2603 ). We fouWe also examined the 4-gene scores in primary and bone, brain, and lung metastasis tumors of the GSE110590 cohort . Scores p ≤ 0.033). For lung metastasis, the association was present in all three cohorts , mid (n = 14), and bad (n = 10). Indeed, the 4-gene score of tumors was significantly reduced after NAC for patients who had a good response  regimen consisting of anthracycline and taxane , and theresponse A. Such aresponse A 37]..n = 8), It is well known that highly proliferative, aggressive breast cancers are more likely to respond to NAC. We, therefore, speculated that a high 4-gene score prior to NAC will be associated with more cancer aggressiveness and thus a better response to NAC. To examine this, we analyzed each cancer subtype independently because it is well known that the therapeutic effect differs among subtypes ,39,40. IPD1, (programmed death 1), and PDL1 (programmed death ligand 1), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), LAG3 (lymphocyte activation gene 3), IDO1/2 (indoleamine dioxygenase 1/2) (HLA (human leukocyte antigen), TIGIT (tyrosine-based inhibitory motif domain), TIM-3 (T cell immunoglobulin domain and mucin domain 3), and VISTA (V-domain immunoglobulin-containing suppressor of T cell activation)  C. These ivation) . To thisThe aim of our study was to develop a prognostic biomarker for occurrence of distant metastasis in breast cancer based on gene expression profiles of bulk tumors. To identify genes for such a biomarker, we compared the gene expression of poorly metastatic MDA-MB-231 human breast cancer cell line and its variant, LM2-4, which was selected in vivo after multiple rounds of implantation/resection for higher tendency for spontaneous metastatic spread ,23. The tumor expression of 4 genes that were upregulated in LM2-4 was identified to have a strong association with DFS in the TCGA cohort. We used these genes to develop a 4-gene score and found that the score was prognostic of survival for the TCGA patients, with higher scores associated with worse survival. These results were validated with another large cohort, METABRIC. Although we could not detect a clear difference in the performance in the TNBC and HER2+ breast cancers, the score was a prognostic biomarker for HR+/HER2- breast cancer patients. The result suggests that the score was not reflected by the aggressiveness determined by subtype alone. Furthermore, the prognostic performance of the 4-gene score was good for late-stage cancer. Interestingly, none of the genes that were included in the 4-gene score was previously considered to be a major player in breast cancer metastasis. PGF  is a member of the pro-angiogenic vascular endothelial growth factor (VEGF) family, and its overexpression has been linked to pathological angiogenesis . Thus, PDownstream of tyrosine kinase (DOK) proteins are adaptor proteins that are widely implicated in the regulation of cell growth, differentiation, and death, as well as cell motility . DOK4 isHCCS (holocytochrome c synthase) is an enzyme that is required for mitochondrial cytochrome c maturation and therefore respiration . HCCS haSHCBP1 (SHC SH2-binding protein 1), a member of Src homolog and collagen homolog family, has been reported to be over-expressed in several malignancies including breast cancer. SHCBP1 regulates breast cancer cell proliferation through apoptosis and cell cycle regulation . SHCBP1 Gene expression measurements of primary breast cancer tumors have been investigated as predictive as well as prognostic biomarkers in hundreds of studies. At least 5 tumor gene expression-based biomarker assays are in clinical use. MammaPrint, the first such assay to be developed , relies Gene set enrichment analysis suggests that tumors with high 4-gene scores are more aggressive, as observed from their higher expression of genes involved in cells. On the other hand, tumors with low scores have a higher expression of genes associated with estrogen response. This is in agreement with the observation that HR+/HER2− tumors have low scores. The 4-gene score was prognostic for the development of distant metastasis. The scores of tumors that spread to the brain or lung were higher compared to those that did not metastasize or did so only to bone. Additionally, score values of brain or lung metastases were significantly higher compared to pre-metastatic primary tumors or bone metastases. The lack of predictive value of the score for bone metastasis may be because DFS was used for the selection of the genes to develop the 4-gene score, and metastases to bone are not as lethal as those to the brain or lung. We used three different cohorts and found that high score values of primary tumors were associated with shorter time-intervals for occurrence of metastasis, especially to the brain or lung.The 4-gene scores of tumors were significantly reduced by chemotherapy or hormonal therapy when clinical response to treatment was good. Interestingly, the pCR rate for NAC among high-score tumors was significantly higher than low-score tumors. This observation was noted in two independent cohorts for the HR+/HER2− subtype. These results suggest that the 4-gene score may be useful for the patient selection for neoadjuvant treatment for HR+/HER2− breast cancer patients. The HR+/HER2− subtype accounts for approximately 70% of advanced breast cancer and is thus responsible for most of the deaths from the disease. It is believed that response to NAC, one of the main treatments for breast cancer, is relatively poor for HR+/HER2− breast cancer due to its low aggressiveness. While many efforts are ongoing to circumvent resistance mechanisms with a few strategies already incorporated into clinical practice, their effectiveness is still limited. Therefore, the ability to predict NAC responsiveness in HR+/HER2- breast cancer with a biomarker like our 4-gene score can be of significant utility.Immune checkpoint inhibitors, such as those targeting the PD1/PDL1 axis, have shown modest activity as monotherapy in advanced breast cancer. Atezolizumab, an immune checkpoint inhibitor, combined with nab-paclitaxel was recently approved for breast cancer and now is in clinical use . HoweverOur study is not free from limitations. To assess the prognostic or predictive value of the 4-gene score, we used its within-cohort top one-third percentile value to stratify patients. Our study does not establish an absolute value for interpretation of the score. This is because we utilized data from a variety of studies for our analyses, and different gene expression and data processing methods were used in these studies. Further work is required to refine our scoring method  so that the score can be interpreted by absolute value and used on a single-sample basis. Both hybridization microarray and RNA sequencing methods were utilized for gene expression profiling of tumors of the cohorts that we examined. How the gene expression measurement platform affects the 4-gene score value is unknown at this point and likely requires further investigation. Another limitation is that our 4-gene score was generated using only the upregulated genes in LM2-4 compared to parental MDA-MB-231 cells and disregarded the downregulated genes. Commonly, genes significantly downregulated are as informative as upregulated genes for building a score and omitting them may have missed novel biomarkers. Therefore, it is possible that a score that includes the downregulated genes may have better performance than the 4-gene score and development of such a score may become one of the future studies.LUC+ variant of MDA-MB-231 [2. Identity of the cell lines was verified by simple tandem repeat profiling.The MDA-MB-231 human breast cancer cell line (ExPASy Cellosaurus RRID: CVCL_0062) was acquired from ATCC, Manassas, VA. The LM2-4A-MB-231  was kind2-transformed for further analyses.Each of the two cell lines was examined in triplicate. Both lines were cultured concurrently, and harvested at sub-confluence for RNA isolation on three different days. An affinity spin-column-based total RNA purification kit with a DNAse treatment step was used . RNA preparations were quantified with TapeStation RNA ScreenTape . RNA integrity number values were above 8.0. Sequencing libraries were prepared from 1 µg RNA using reagents and protocols provided in TruSeq Stranded Total RNA Library Prep Gold kit . Ribosomal RNA depletion and 15 PCR cycles were employed during library preparation to increase the cDNA molarity. TapeStation D1000 ScreenTape (Agilent) and Library Quantification Kit  assays were used to confirm good quality of libraries. All six libraries were sequenced together on an Illumina HiSeq 2500 instrument using HiSeq Rapid Cluster Kit v2―Paired-End and Rapid SBS Kit v2 reagents to obtain paired reads of 100 bases. Casava software  was used to demultiplex sequencing data. An average of 100 million sequence read-pairs were obtained for each sample. Raw read data were filtered to remove adapters and poor-quality segments, and then mapped in a splicing-aware manner to the hg38 human reference with TopHat2 . TopHat22 fold-change (FC) >1.2 and false discovery rate (FDR) <0.05 after adjustment for multi-testing by Benjamini–Hochberg method were considered as differentially expressed. For multiple testing adjustments, the Benjamini–Hochberg method was used to calculate the FDR, where the FDR < 0.05 was chosen as cut-off to identify the candidate genes. For gene set enrichment analysis, GSEA [http://software.broadinstitute.org/gsea/index.jsp) and mSigDb [p value threshold of 0.05 and a FDR of 0.25, as recommended by the GSEA software, was used to deem significance.For differential gene expression analyses of the cell lines, the DESeq2 Bioconductor package for R was used. Genes with expression changes of absolute logis, GSEA  softwared mSigDb  Hallmarkn = 286) [n = 82) [n = 16) [n = 204) [n = 61) [n = 508) [n = 33) [n = 11) [2-transformed data was used for further analyses.Tumor gene expression and clinical data for 1094 patients of the TCGA breast cancer (BRCA) project  were obtn = 286) , Minn et[n = 82) , Siegel [n = 16) , Bos et n = 204) , Iwamoto[n = 61) , Symmansn = 508) , Vera-Ra[n = 33) , and Mas[n = 11) , from thDOK4, HCCS, PGF, and SHCBP1 had HR ≥ 1.2 with p ≤ 0.02 in this analysis. Tumor expression of these four genes and DFS was then analyzed with a multivariate Cox regression model. The formula for calculating a 4-gene score using gene expression and Cox model regression coefficient values was thus established as:1.355 × (expressionDOK4) + 1.641 × (expressionHCCS) + 1.345 × (expressionPGF) + 1.232 × (expressionSHCBP1). Within any cohort, the high/low cutoff for the 4-gene score was defined as the top-third versus the bottom two-thirds throughout our work. Uniform cut-off values could not be used for all cohorts because of differences in platforms and data processing methods for generation of gene expression data.In order to determine which gene’s expression may be an independent prognostic marker for DFS, univariate Cox regression analysis was performed using the TCGA cohort. Among the 297 genes that were differentially upregulated in LM2-4 compared to parental MDA-MB-231 cells, http:///www.r-project.org/) or Microsoft Excel . Unless noted otherwise, Kaplan–Meier method with logrank test was used for survival analysis. A cut-off of 0.05 for p value was used to judge significance in statistical tests, which were always two-tailed and assumed equal group variances. All statistical analyses and data plotting were performed using R (In conclusion, by combining gene expression data of cancer cell lines with publicly available clinical and gene expression data of multiple published studies, we were able to establish a novel 4-gene score for breast cancer tumors. Multivariate and stratified analyses showed that the score is useful as a biomarker independent of clinical and pathological factors. The score may have utility for predicting survival, recurrence, and distant metastasis, as well as response to neoadjuvant therapy and possibly immunotherapy in breast cancer.


The BR control was studied using phenylephrine (Phe) and sodium nitroprusside (SNP) through sigmoidal analysis. The autonomic control of the heart was estimated using heart rate variability (HRV) analysis in frequency domain. Additionally, to determine the maximum sympathetic and parasympathetic activation of BR, spectral non-stationary method analysis, during Phe (0.05 μg/mL) and SNP administration (0.10 μg/mL) were used. Compared to Sea-Level condition, the High-Altitude group displayed parasympathetic withdrawal  and sympathoexcitation . Regarding to BR modulation, rats showed a significant decrease (p < 0.05) of curvature and parasympathetic bradycardic responses to Phe, without significant differences in sympathetic tachycardic responses to SNP after High-Altitude exposure. In addition, the non-stationary analysis of HRV showed a reduction of parasympathetic activation (Phe) in the High-Altitude group. Our results suggest that acute exposure to High-Altitude produces an autonomic and BR control impairment, characterized by parasympathetic withdrawal after 24 h of high-altitude exposure.Baroreflex (BR) control is critically dependent of sympathetic and parasympathetic modulation. It has been documented that during acute hypobaric hypoxia there is a BR control impairment, however, the effect of a natural hypoxic environment on BR function is limited and controversial. Therefore, the aim of this study was to determine the effect of acute High-Altitude exposure on sympathetic/parasympathetic modulation of BR control in normal rats. Male Sprague Dawley rats were randomly allocated into Sea-Level ( The cardiac baroreflex (BR) function governs short-term fluctuations of blood pressure and, therefore, plays an important role in the regulation of several homeostatic functions . The maiAmong the aforementioned conditions, High-Altitude is amongst the most inhospitable environments on earth and it has been demonstrated that exposure to hypobaric hypoxia is strongly related to impairment of autonomic control . AlthougTwelve male Sprague-Dawley rats were used in these experiments. All surgical procedures and protocols used, were in accordance with guidelines of the American Physiological Society and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Antofagasta Scientific Research Ethical Committee (CEIC-210/2019).n = 12) were housed in individual cages with a 12/12-h light/dark schedule and were allowed free access to food and water. The rats were randomly allocated into Sea-Level group (n = 7) and to High-Altitude group (n = 5). Sea-level rats were subjected to catheterization surgery according to the method of i.v.) and after 30 min of recovery, 8 boluses of sodium nitroprusside to decrease BP were injected (i.v.). The second series of rats (High-Altitude group) ascended at 3,270 m above sea level  in a costume made mobile laboratory and after 24 h, the catheterization surgery was performed. Similar to the first animal series (Sea-Level group), 8 h after  using ketamine  plus xylazine  , were performed to measure BP and for drugs administration. The rats were anesthetized ( States) . A midli States) . The BP  States) .The BR was evaluated by repeated bolus injections (0.1 ml) of graded doses of sodium nitroprusside  and phenylephrine . These drugs were used to induce a decrease or increase in BP, respectively. Sodium nitroprusside and phenylephrine injections were given in a random order and subsequent injections were not given until the recorded parameters had returned to pre-injection levels. The cardiac BR function was analyzed using a logistic regression over the entire pressure range . Data waTo determine whether the effects of High-Altitude in BR control could be associated to differences in BP stimulus, we constructed a dose-response curve for SNP and Phe. We used 8 doses of SNP  and 8 doses of Phe . The curve was constructed using the logarithm of different doses. The responses were estimated using the delta of MABP (ΔMABP) from previous baseline measurements.HRV): 0.04–0.6 Hz and high frequency (HFHRV) 0.6–2.4 Hz . Analysis was performed within a 10 min window. This analysis was performed in LabChart 7.3.8 HRV module software . In addition, to estimate the autonomic contribution on BR function, spectral non-stationary analysis was used (2-s resolution). The HFHRV component (0.6–2.4 Hz) was used as an indicator of parasympathetic modulation. This analysis was performed with Kubios HRV Premium Software V 3.1 .Heart rate variability (HRV) was used as an indirect measurement of autonomic balance of the heart . The fir6–2.4 Hz . Additiop < 0.05 was considered statistically significant. Statistical analyses were performed by GraphPad Prism 8.0 .Data were expressed as mean ± standard error of the mean. All data were subjected to Shapiro-Wilk normality test. The unpaired t-test at two tails was employed to compare the differences between groups. Baseline physiological variables for both groups are shown in HRV component was significantly increased (p < 0.05) from Sea-Level (42.12 ± 7.44 n.u.) to High-Altitude (60.55 ± 4.47 n.u.) (HRV component was significantly reduced (p < 0.05) from Sea-Level (39.37 ± 4.44 n.u.) to High-Altitude (57.82 ± 7.43 n.u.) (HRV ratio was significantly increased (p < 0.05) at High-Altitude compared to Sea-Level  , while t43 n.u.) . Consequp < 0.05) (p < 0.05) after acute High-Altitude exposure compared to Sea-Level. However, maximum tachycardic response to SNP, range, slope, midpoint of BP, lower plateau and upper plateau of BR analysis, were not significantly different between Sea-Level and High-Altitude groups (The BR parameters after High-Altitude exposure are shown in  < 0.05) . Indeed, < 0.05) . Moreove < 0.05)  and maxi < 0.05)  were sige groups .p < 0.05) (p < 0.05) (p < 0.05) between resting condition compared to SNP administration  , however < 0.05) . LF and stration . Regardistration . VLF, LFstration .R2  after acute High-Altitude exposure there is a cardiac autonomic control impairment; (ii) exposure to High-Altitude produces a deterioration of cardiac BR control  in normal rats; and (iii) there is a reduced activation of parasympathetic drive during acute High-Altitude exposure in normal rats. The present results suggest that acute exposure to high-altitude produces an autonomic control impairment and a deterioration of BR function, mainly characterized by parasympathetic withdrawal after 24 h of high-altitude exposure.Estimations of the World Health Organization  indicateIt has been proposed that acute hypoxia is a potent activator of sympathetic drive in a time dependent manner . MoreoveIn addition, despite that in our experiments we did not evaluate norepinephrine release, our results showed that BR maximum tachycardic response was not different between sea level and high-altitude exposure, suggesting that the sympathetic drive during high-altitude could not be related to BR control, if not to an interaction between peripheral chemoreflex activation and baroreflex control impairment, triggering parasympathetic withdrawal . NeverthIn addition, from a practical perspective, our work could be applied to people exposed to “live low – work high” conditions. In several countries,  the growth domestic product is critically dependent of bigger mining whose operations are mostly at high-altitude. Additionally, Chile is a country with an important number of astronomers  exposed to the aforementioned “live low – work high” conditions. Further, in several sports, competitions are celebrated at high-altitude . Therefore, our results could transfer to people which are subjected to high-altitude exposure with a focus on improving baroreflex control and parasympathetic modulation of the heart, which could improve the functional capacity at high-altitude.Regarding some potential limitations of our study, we did not use the same animals at Sea-Level and at High-Altitude conditions, which could mask some physiological responses to High-Altitude exposure. However, our results showed that after acute High-Altitude exposure our animals displayed several characteristics of hypobaric hypoxic exposure. In addition, our experiments were limited to 24 h post-High-Altitude condition. Thus, it is not possible to infer the long-term effects of High-Altitude exposure on cardiac BR control from our study. Moreover, we did not determine the contribution of ventilatory chemoreflex control, which could be related to BR control impairment after 24 h of High-Altitude exposure . In addiCardiac autonomic dysfunction and BR control impairment occur after 24 h  of High-Altitude exposure. In addition, the parasympathetic activation was decreased after high-altitude exposure. Therefore, the short-term exposure to high-altitude produces an autonomic control impairment and BR dysfunction, which could be more related to parasympathetic modulation of the heart rather than sympathetic drive.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by University of Antofagasta Scientific Research Ethical Committee (CEIC-210/2019).AB, AA-Á, MV-M, RR-C, MI, MR, RD and DA designed the work and contributed to analysis, interpreted the the data, and drafted the work. AB and DA performed the data collection and analysis and contributed to the concept of the project and experimental design. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Considerable disagreement exists on the linearity of the development of standing balance in children. This study aimed to use different traditional and nonlinear methods to investigate age-related changes in standing balance in preschoolers. A sample of 118 preschoolers took part in this study. A force platform was used to record the center of pressure during standing balance over 15 s in three conditions: eyes open, eyes closed, and/or head extended backward. Detrended fluctuation analysis (DFA), recurrence quantification analysis (RQA), and traditional measures were used to evaluate standing balance. The main results are as follows: (1) Higher range and SD in the anterior-posterior (AP) direction were observed for 5-year-old than for 4-year-old children, while higher DFA coefficient  and higher determinism and laminarity in the AP direction were found for 5-year-old children compared to 3- and 4-year-old children; and (2) as sensory conditions became more challenging, all traditional measures increased and DFA coefficients  decreased in the AP and mediolateral directions, while determinism and laminarity significantly declined in the AP direction. In conclusion, although increased postural sway, 5-year-old preschool children’s balance performance improved, and their control strategy changed significantly compared with the younger preschoolers. Sensory perturbation (eye closure and/or head extension) changed preschoolers’ balance performance and control strategy. Moreover, both traditional and nonlinear methods provided complementary information on the control of standing balance in preschoolers. Maintaining standing balance is a complex sensorimotor process. It involves multiple sensory systems and actions of muscles distributed over the whole body. A deficit in any sensory systems or integration of multisensory information can affect standing balance . The conThe most common method for assessing standing balance is the postural sway’s characterization by measuring the center of pressure (COP) displacements . COP sigIn previous studies, most authors investigated the age-related changes in standing balance in preschoolers under challenging conditions by perturbating the sensory inputs of vision and/or proprioception ,b. EspecThus, the purpose of the current study was to investigate how age and sensory perturbation affect the control of standing balance for preschool children on a firm surface using traditional and nonlinear methods. The hypotheses were as follows: (1) The balance performance and control strategy of 5-year-old preschool children’s standing balance would change significantly compared with the younger preschoolers; (2) sensory perturbations (eye closure and/or head extension) would change the control of preschoolers’ standing balance accordingly; and (3) both traditional and nonlinear methods may discriminate the age-related changes in standing balance in preschoolers, and nonlinear methods may provide different information about the effect of age on standing balance.n = 40), 4-year-old children (n = 39), and 5-year-old children (n = 39). The parents or guardians of children provided written consent before any measurements. A questionnaire was also completed by the parents or guardians to identify any presence among participants of developmental problems or interest in cooperation, which were all considered exclusion criteria. Participants were recruited from one of the regular preschools in Hangzhou, China. This study was approved by the local ethical committee of Zhejiang University (issued no. 2020-003) and conformed to the Declaration of Helsinki.A cross-sectional study was performed in a sample of 118 preschool children. They were grouped according to chronological age: 3-year-old children (n = 36), 4-year-old children (n = 38), and 5-year-old children (n = 39). p < 0.01 for height and body mass).Among the 3-year-old children, 90% completed all conditions (four children were excluded because of test failure). Among the 4-year-old children, 97.44% completed all conditions (only one child was excluded because of test failure). Among the 5-year-old children, 100% were able to complete all conditions. In total, the final sample of 113 children are presented, which include 3-year-old children  with feet together  for 15 sDemographic data  were reported. All signals from the force platform were processed offline using MATLAB software . The COP positions were calculated from the ground reaction forces and moments of force and then filtered using a 20 Hz low-pass, 2nd order, zero-lag Butterworth filter. Furthermore, the mean of the filtered data was removed. The COP displacements were subsequently analyzed using traditional and nonlinear methods in the anterior-posterior (AP) and mediolateral (ML) directions. Traditional methods included the range, SD, sway mean velocity, sway path length, and sway area, which quantified the postural sway ,b. NonliThe range is the distance between the maximum and minimum COP displacement in the AP and ML directions, representing the entire trial’s postural sway. In general, the greater the range, the worse the postural stability . BecauseN = number of samples, F = sampling frequency.where x(i) and y(i) are the COP displacements in the ML and AP directions, respectively, Sway area quantifies 85% of the total area covered in the ML and AP directions using an ellipse to fit the COP data, which is considered an index of overall postural performance . Figure 1) in general greater than 1.5, indicating that a persistent pattern of COP sway. The second slope (DFA2) in general smaller than 1.5, indicating that an anti-persistent pattern of COP sway. Higher DFA values indicate smoother and more persistent behavior at short (DFA1) and long (DFA2) time scales, while lower DFA values indicate less smooth and more anti-persistent behavior at short (DFA1) and long (DFA2) time scales  is the original COP signal at time t, where The fluctuation of the integrated COP signal is calculated as: n. The slopes of the fitted lines of the log-log plot at short and long time scales are the DFA coefficients (DFA1 and DFA2).where Recurrence quantification analysis is a tool for studying the dynamics of a signal. It is based on the construction of a recurrence plot (RP) from which quantitative measures are extracted. The time delay was calculated using the mutual information method, and the embedded dimension was determined using false nearest neighbor analysis. We calculated Determinism (%DET) and Laminarity (%LAM) of the COP signal as follows:Recurrence plot is briefly defined as ε is a predefined threshold and m-dimension phase space;where l of diagonal structures, and lN is the absolute number of diagonal lines;where l of vertical structures.where 1 and DFA2), determinism (%DET), and laminarity (%LAM) in the AP and ML directions.The following measures were selected for analysis. Traditional measures included amplitude (Range), SD, and sway mean velocity (MV) in the AP and ML directions; sway path length (Path) and sway area (Area) were also included. Nonlinear measures included DFA coefficients (DFAp > 0.05). A mixed repeated-measures ANOVA with between-subject factors (age) and within-subject factors (condition) was conducted to assess the effects of age and condition on all the measures. Significant interactions were explored further using simple effects analyses and performed with Bonferroni post hoc tests. Greenhouse-Geisser corrections were applied for circumstances in which sphericity could not be assumed . The significance level was set as p < 0.05 with two-tailed. Effect size values (ηp2) were reported for ANOVA. All statistical testing was conducted using SPSS .Each measure’s distribution of normality was tested  and SD_ap (p = 0.03) were significantly higher for the 5-year-old children than for the 4-year-old children. Effect sizes were small to medium, ranging from 0.058 to 0.060 . Effect sizes were large, ranging from 0.354 to 0.600 , DFA1_ap was significantly higher for the 5-year-old children than 3- (p < 0.01) and 4-year-old children (p = 0.04), %DET_ap was significantly higher for the 5-year-old children than 3- (p < 0.01) and 4-year-old children (p = 0.02), and %LAM_ap was significantly higher for the 5-year-old children than 3- (p < 0.01) and 4-year-old children (p < 0.01). Medium to large effect sizes were found for DFA1_ap, %DET_ap, and %LAM_ap, ranging from 0.093 to 0.153 .to 0.153 . No age-1_ml, DFA1_ap, DFA2_ml, DFA2_ap, %DET_ap, and %LAM_ap. Post hoc tests revealed that %DET_ap and %LAM_ap (p > 0.05) were not statistically different for the EO and EC. %DET_ap was significantly lower for the ECHB than EO (p < 0.01) and EC (p < 0.01), and %LAM_ap was significantly lower for the ECHB than EO (p < 0.01) and EC (p < 0.01). DFA1_ap was significantly lower for the ECHB than EO (p < 0.01) and EC (p < 0.01), DFA2_ml was significantly lower for the ECHB than EO (p < 0.01) and EC (p < 0.01), DFA2_ap was significantly lower for the ECHB than EO (p < 0.01) and EC (p < 0.01), while DFA1_ml (p < 0.01) significantly decreased as conditions became more challenging. Medium to large effect sizes were found for DFA1_ml, DFA1_ap, DFA2_ml, DFA2_ap, %DET_ap, and %LAM_ap, ranging from 0.120 to 0.172  5-year-old children showed more postural sway in the AP direction than 4-year-old children; (2) 5-year-old children showed decreased variability and more intermittent in the AP direction than 3- and 4-year-old children; (3) standing balance in the ML direction was the same for 3- to 5-year-old children; (4) as the sensory conditions became more challenging, the amount and variability of postural sway increased, while intermittency decreased; and (5) traditional and nonlinear methods provide complementary information for evaluating standing balance in preschoolers. These results are discussed below.According to traditional methods, the age-related difference of standing balance in preschoolers was only found between the 4- and 5-year-old children. This difference was shown with higher Range_ap and SD_ap in the AP direction for the 5-year-old children, consistent with the previous study . MV_ap, 1_ap, consistent with the previous study  in the AP and ML directions. These results indicated that less persistent COP sway at shorter time scales and more anti-persistent pattern of COP sway at longer time scales as conditions became more challenging. Also, there was no difference between EC and EO for %DET in the AP direction, consistent with the previous study  changed preschoolers’ balance performance and control strategy. Moreover, both traditional and nonlinear methods provided complementary information on the control of standing balance in preschoolers.The data presented in this study are available on request from the corresponding author.The studies involving human participants were reviewed and approved by Research Ethics Board of Center for Psychological Sciences at Zhejiang University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ZH contributed to writing the original draft, revising and editing the manuscript, data collection, data analysis, statistics, and data interpretation. YY and AH contributed to the data collection and data analysis. YG contributed to revising and editing the manuscript. JW contributed to conceptualization of the study, data interpretation, and revising and editing the manuscript. All authors approved the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Attenuation correction of PET/MRI is a remaining problem for whole-body PET/MRI. The statistical decomposition algorithm (SDA) is a probabilistic atlas-based method that calculates synthetic CTs from T2-weighted MRI scans. In this study, we evaluated the application of SDA for attenuation correction of PET images in the pelvic region.Twelve patients were retrospectively selected from an ongoing prostate cancer research study. The patients had same-day scans of [11C]acetate PET/MRI and CT. The CT images were non-rigidly registered to the PET/MRI geometry, and PET images were reconstructed with attenuation correction employing CT, SDA-generated CT, and the built-in Dixon sequence-based method of the scanner. The PET images reconstructed using CT-based attenuation correction were used as ground truth.The mean whole-image PET uptake error was reduced from − 5.4% for Dixon-PET to − 0.9% for SDA-PET. The prostate standardized uptake value (SUV) quantification error was significantly reduced from − 5.6% for Dixon-PET to − 2.3% for SDA-PET.Attenuation correction with SDA improves quantification of PET/MR images in the pelvic region compared to the Dixon-based method. Correct quantification of radiotracer activity in PET is of importance when monitoring cancer treatment response. PET quantification relies on accurate attenuation measures, which is a remaining challenge with PET/MRI. In PET/CT systems, the attenuation map for 511 keV PET photons is calculated from the CT image, typically with a bilinear equation acetate PET/MRI images. The attenuation corrected PET images are validated against AC based on CT and the method is also compared with the Dixon-based method on the PET/MRI scanner.In this project, we evaluate a statistical decomposition algorithm (SDA)  for calcClinicalTrials.gov ID NCT01962324). All patients underwent [11C]acetate PET/MRI and CT scans on the same day. Patients were included if they had a radiotherapy treatment planning with delineated regions for prostate, and one hotspot volume within the prostate, and saved PET raw data. Patients were excluded if they had implants in the scanned field of view (FOV).Twelve prostate cancer patients  were selected retrospectively from a clinical research study , commencing 12.6 ± 3.3 min after injection of a 431 ± 53-MBq bolus dose of [11C]acetate.2, slice thickness 5.2 mm with a 2.6 mm overlap, and 120 slices in total. T2-weighted scans  were acquired with repetition time 15 s, echo time varying between 96.2 and 102.8, flip angle 130°, matrix size 1024 × 1024, pixel size 0.44 × 0.44 mm2, slice thickness 2.5 mm, and 98–112 slices depending on patient anatomy.A 2-echo Dixon T1-weighted MRI sequence (Liver Acquisition with Volume Acceleration: LAVA-Flex) was acquired for built-in AC based on segmentation of fat and water with in-phase and out-phase images. The following parameters were used: repetition time 4.05 ms, echo time 1.12 ms and 2.23 ms, flip angle 5°, matrix size 258 × 258, pixel size 1.95 × 1.95 mm2, and slice thickness 2 mm.CT images were acquired on a Brilliance Big Bore CT  with tube voltage 120 kV, automatic exposure (204 ± 94 mAs), matrix size 512 × 512, pixel size 1.014 × 1.014 mmThe SDA (15) can be described as an atlas-based technique where tissues are registered separately. It takes a T2-weighted MRI scan as input and returns the most probable CT representation as output. The SDA was utilized from the online software MriPlanner . The algorithm uses a template database consisting of 15 MRI and CT scans from the same individuals, pairwise registered using non-rigid registration, with delineated structures of the prostate, bladder, colon, bones, and subcutaneous fat.The SDA can be divided into three steps. The first step is automated segmentation of the incoming MRI. In this step, the template MRIs are registered to the incoming MRI, and the deformation fields are applied on the template segmentations to generate a set of segmentation candidates. The final segmentation is calculated using a weighted voting-method based on machine learning  which prThe CT scans were adapted to the PET/MRI space with registrations performed with the Elastix toolbox  implemenThe outermost part of the skin was barely visible on the T2-weighted scans, and the out-of-phase images displayed blurred contours, which creates uncertainties in patient contour. If not corrected for, these uncertainties can propagate into the registered CT, since the T2-weighed scans are used for registration. The uncertainties were eliminated by adapting the outer contour for CT and SDA-CT to the same contour as the Dixon-CT. This was achieved by replacing voxel-values in CT and SDA-CT outside the outer contour of the Dixon-CT with the CT-value of air (HU − 1000), and add tissue with the CT value of water (0 HU) where the Dixon-CT was larger than the CT or SDA-CT.PET images were reconstructed with AC based on each of the three imaging methods  employing the PET recon toolbox , version MP26, release 30.4. Time-of-flight ordered-subset expectation-maximization (TOF-OSEM) was chosen to reconstruct the data according to our clinical protocol: 2 iterations, 28 subsets, 600 mm transaxial FOV, 25 cm axial FOV, matrix size 192 × 192, 89 slices, pixel size 3.125 × 3.125 mm, and slice thickness 2.78 mm. Attenuation information for CT and SDA-CT was fed into the PET recon toolbox as CT images, which were re-calculated into AC-maps (CT-AC and SDA-AC). Dixon-based AC maps (Dixon-AC) were also generated with the PET recon toolbox as a part of the reconstruction process. PET reconstructions were performed with truncation completion activated, which means that missing parts of the patient’s outer contour were estimated from the PET images .−1 were segmented as bone. Images of percentage differences were calculated. From these difference images, values of mean error, mean absolute error (MAE), standard deviation (SD), and root mean square error (RMSE) were calculated for the whole image and separately for the above-segmented bone and soft tissue. The same statistical measures were calculated comparing Dixon-AC and SDA-AC with CT-AC as ground truth.The accuracy of Dixon-PET and SDA-PET were calculated over the whole FOV, using only the slices that were present in all AC-maps. CT-PET was considered as the ground truth. Voxels were segmented into the bone and soft tissue based on the attenuation map from the CT, where all voxels with attenuation coefficients higher than 0.102 cmFor visualization of the PET and AC error distribution, error maps averaged over all patients were calculated in a common space. One patient was manually selected as reference geometry, where the selection aimed for an average patient size. All error maps were then subject to a non-rigid registration to the geometry of this reference patient. The T2-weighed MRI of each patient was registered to the T2-weighted MRI of the reference patient with the same method as described for the CT to MRI registration in the data pre-processing section, and the deformations were applied on the error maps for each patient. The registered error maps were used for calculation of a mean error-map for all patients.Lesion analyses were performed with volumes of interest (VOIs) drawn by specialists in oncology as a part of radiotherapy treatment planning. The prostate and a prostate-hotspot were compared regarding mean standardized uptake value (SUV) within the VOI. SUV error (%ΔSUV) was calculated for Dixon-PET and SDA-PET as a percentage difference relative to CT-PET. Statistical significance was tested with the Wilcoxon signed-rank test.The Dixon-PET error map Fig.  demonstrThe absence of bone information is also very clear in the error maps for attenuation coefficients Fig. , where aHistograms of PET image voxel errors Fig.  confirm For AC-maps, soft tissue histograms Fig.  are narrp = 0.0024). For hotspot lesions, the mean values were − 5.9% (Dixon) and − 2.3% (SDA), also with a significant difference between methods (p = 0.00098).The lesions in this study consisted of the prostate , including also one hotspot (3 ± 4.7 ml), which can be seen in Fig. We have evaluated SDA-generated synthetic CT (sCT) images for attenuation corrected PET reconstructions in the pelvic region and compared that with attenuation corrected PET reconstructions generated with CT images. The SDA calculates sCTs from T2-weighted MRI sequences with a probabilistic atlas-based technique, where the most probable CT representation for a given T2 image is calculated. The results are promising despite the fact that other atlas methods have had limited success in the pelvic region .The error maps in Fig. −1. The threshold was chosen to minimize the risk of misclassifying bone as soft tissue, and a higher threshold can be expected to yield higher errors in soft tissue since the largest errors are close to bone.Three previous studies have presented attenuation maps from MRI using neural networks, with ZTE and Dixon sequences as input in a method called ZeDD , with onThe main benefit of the SDA-method is that it only relies on T2-weighted images, which are commonly acquired for diagnostic purposes. This means that the scanning time does not have to be prolonged, since additional sequences are not required. This is also a benefit of the method by Bradshaw et al.  with onlStudies that compare sCTs with CTs need to rely on registration, which entails some uncertainties also in this study. First, the registration in itself leads to uncertainties, even if the registrations were visually assessed to be accurate in the studied region. The largest registration uncertainties were found in the patient outer contour, where we chose to adapt all AC-maps to the outer contour for the Dixon method. This was done to minimize the uncertainties in the registration of the CT which was used as a gold standard. Another shortcoming related to registration is that bowel air can move between examinations. To minimize the effect, the voxel values of air pockets were replaced with HU 0 for water.The reconstruction of the PET images in this study is conducted according to our clinical protocol; TOF-OSEM with 2 iterations and 28 subsets. It is possible that more iterations could have some effects on the results for the Dixon-PET, where the absence of bone information gives larger errors in the attenuation maps and affects the PET image globally . The numIn this study, the patient group is limited to prostate cancer patients, a male population with a higher age than the average population. This age group can be expected to have lower HU values in bone, and it remains to evaluate if the results of this study are generally applicable to other age groups, and to female patients, or if the SDA algorithm may have to be adapted. Although the SDA captures bone, full HU recovery is not reached. It is hard to speculate about the reasons, but anatomical differences between this patient group and the patients in the template database could possibly be an explanation.We have evaluated a probabilistic atlas method for AC of pelvic PET images in PET/MRI scanners. The evaluated method improves quantification compared to the currently used Dixon-based method.Additional file 1. Parameters


Previous studies have shown that temporal self-construal priming can modulate the empathic neural responses to others’ pain. However, little is known about the influences of the dispositional self-construal on empathic neural responses to others’ pain. The present study aimed to investigate neural correlates that underlie the modulation effect of dispositional self-construal on perception of others’ pain. Event-related potentials were recorded for pictures depicting the hands of strangers in painful or no-painful situations while subjects performed a pain judgment task. The regression analysis on behavioral data showed that the level of interdependent self-construal could positively predict behavioral ratings of perceived pain, but not the self-unpleasantness. The ERP results showed painful stimuli elicited decreased N2 amplitudes and larger P3 amplitudes than those by no-painful stimuli. Moreover, the level of interdependent self-construal (interdependence minus independence scores) could predict the amplitude differences on the P3 component , but not the N2 component: the higher the level of the interdependent self-construal, the larger amplitude differences of P3 to painful stimuli (vs. no-painful stimuli). These findings extended previous studies by showing a clear modulation effect of the dispositional self-construal on empathic neural responses to others’ pain, and that this modulation effect occurred at the late cognitive evaluation stage indexed by the P3 component. Pain empathy refers to the capacity to understand and share others’ pain, as well as the corresponding affective-motivational reactions, which plays an important role in social communication and human interactions . PreviouTaken together, these findings indicate that empathic neural responses are modulated not only by what the pain-perceiver and target person are but also by the relationship between pain-perceiver and target person. Nevertheless, the relationship between the pain-perceiver and the target person is not static and actually depends on how the perceiver views and values this relationship. Self-construal is a construct, which refers to how people view themselves and their relation to others and the social environment . AccordiPrevious ERP and neuroimage studies have shown that temporary shifts in self-construal can modulate the neural activities underlying various cognitive processes such as visual attention, pain perception, self-other processing, and motor-related processing . More reThese studies suggested that empathic neural responses to others’ pain could be modulated by temporary activation of self-construal through a situational prime. However, unlike the temporary situational self-construal, the dispositional self-construal is a stable trait, the formation of which is influenced by long-term cultural experiences . MoreoveIt has been found that the ERP waves elicited by painful vs. no-painful stimulation diverged from the early stage (the N1 and P2 components) to the later stage (the N2 and P3 components) ,b. The NA posteriori statistical power analysis via G∗power was conducted  participated in this study as paid volunteers. onducted , and con2) and no-painful (18.76 Cd/m2) picture were matched, t(38) = 0.024, p = 0.88. The stimuli were presented in the center of a gray background of a 21-inch color monitor, all of which was 7 cm × 5.5 cm (width × height), and subtended a visual angle of 4° × 3.15° at a viewing distance of 100 cm.The stimuli used in this experiment were 40 colorful pictures depicting the hands of strangers in painful or neutral situations, which have been used in previous studies . Twenty Each trial began with a fixation cross at the center of the screen for 200 ms, which was followed by a blank interval that varied randomly from 800 to 1600 ms. Subsequently, the picture was presented lasting for 1000 ms. Participants were instructed to judge each picture as painful or no-painful by their left or right index finger. The assignment of the left or right index finger to the painful and no-painful stimuli was counterbalanced across participants. After the picture disappeared, there was an interval of 1000 ms before the onset of the next trial see . The entAfter the electroencephalography (EEG) recording session, participants were instructed to evaluate the intensity of pain supposedly felt by the model of each picture as well as the degree of their own unpleasantness when they saw each picture. The evaluations were measured using a 6-point scale  with the Face Pain Scale-Revised (FPS-R) adapted from the Faces Pain Scale , which cAt the end, participants completed the 24-item SCS on a seven-point Likert scale  so as to evaluate their interdependence and independence of self-construal . ParticiThe EEG was continuously recorded from 64 scalp electrodes mounted on an elastic cap  in conformity to the extended 10–20 system while being referenced online to the FCz electrode. Vertical and horizontal electro-oculogram (EOG) were recorded from above and below the left eye and at left and right outer canthi, respectively. The gain and dynamic range of the amplifier were 0.1 μV and ±3.28 mV, respectively. The EEG and EOG were amplified with a 0.05–100-Hz bandpass and digitized at a sampling rate of 500 Hz. Impedances of all electrodes were kept below 10 kΩ.The electrophysiological data was processed offline using the Brain Vision Analyzer 2.1 software . It was first re-referenced to the average of the left and right mastoid electrode, and then corrected on the VEOG and HEOG based on the independent component analysis (ICA), high-pass filtered above 0.1 Hz and low-pass filtered below 30 Hz, epoched from 200 ms before onset of the stimulus to 1000 ms after its onset, and corrected with a baseline of mean voltage over the 200 ms pre-stimulus interval. Trials of each condition  without any in which EEG voltages exceeded a threshold of ±80 μV were averaged separately followed by the baseline correction.Previous ERP studies in the domain of pain empathy showed that both painful and no-painful stimuli elicited obvious N1 component between approximately 90 and 130 ms, P2 component between approximately 140 and 200 ms, N2 component between approximately 200 and 280 ms over the frontal-central regions, and P3 component after 380 ms over the central–parietal regions . Thus, at-test. The p-values of all analyses were corrected according to Greenhouse–Geisser if violation of sphericity existed. Partial eta-squared  was conducted on the mean amplitudes of the P2 and N2 components, with the pain  and electrode  as within-subject factors. Moreover, in order to investigate the timing features of the P3 component, we added timing  as another factor and thus a three-way repeated-measures ANOVA was conducted on the mean amplitudes of the P3 component. Both accuracy and reaction time were subject to a paired-samples t(29) = −1.94, p > 0.05]. Yet participants responded faster to neutral than to painful stimuli . In addition, both the degree of perceived pain and self-unpleasantness were higher in painful than in no-painful stimulus conditions (p < 0.001).The results showed that response accuracy between painful and no-painful condition did not show significant difference .Moreover, we conducted a regression analysis to examine the relationship between the level of interdependent self-construal (interdependence minus independence scores) and rating scores of perceived pain and self-unpleasantness (painful minus no-painful stimulus conditions). The mean score for interdependent self-construal (interdependence minus independence scores) was 8.27 (±11.41 SD). The rating scores of perceived pain for painful and no-painful stimuli were 4.44 (±0.98 SD) and 1.13 (±0.24 SD), respectively. The rating scores of self-unpleasantness for painful and no-painful stimuli were 3.99 (±1.01 SD) and 1.12 (±0.21 SD), respectively. A linear regression revealed a significant effect of the interdependence on perceived pain  see . MoreoveF = 11.63, p < 0.001, F = 59.43, p < 0.001, F = 96.09, p < 0.001, F = 56.34, p < 0.001, F = 8.16, p = 0.001, p < 0.05). However, no such interaction effects were observed during 400–500 and 500–600 ms  = 2.6, FDR-corrected p = 0.035, see R2 = 0.04, t(28) = −1.12, FDR-corrected p = 0.32] and P3 at 300–400 ms  and 500–600 ms .In order to examine whether the level of dispositional self-construal could modulate the neural empathic responses to pain, we conducted a regression analysis, with the level of interdependent self-construal (interdependence minus independence scores) as the independent variable, and the N2 and P3 amplitude differences (painful minus no-painful stimulus conditions) as the dependent variables. As the pain effects were more obvious over frontal electrodes during the N2 period and 300–400 ms, mean amplitude differences averaged across these electrodes were used for the regression analysis. As for the 400–500- and 500–600-ms intervals, mean amplitude differences averaged across all electrodes were used because there were no significant pain by electrode interactions effect observed during these time intervals. The linear regression revealed a significant effect of the interdependence on the amplitude difference of P3 at 400–500 ms [The present study examined the modulation effect of the dispositional self-construal on neural empathic responses to pain and further determined whether the modulation effect occurred during the early automatic emotional sharing stage, the later attention orienting stage, or the late cognitive evaluation stage. The findings showed that painful stimuli elicited decreased N2 amplitudes and larger P3 amplitudes than those by no-painful stimuli. Moreover, the level of interdependent self-construal positively correlated with the amplitude difference of P3, but not the N2. The behavioral data also showed that the level of interdependent self-construal could positively predict behavioral ratings of perceived pain, but not self-unpleasantness. These findings suggested that the modulation effect of the dispositional self-construal on neural empathic responses to others’ pain occurred at the later cognitive evaluation stage indexed by the P3 component.The present study did not show differences (pain vs. non-painful) for the early N1 and P2 components, which was inconsistent with previous studies showing significant main effects of pain on the N1 or P2 components . These iThe N2 component was considered a “semiautomatic” component, reflecting the final stage of automatic attention-related neural mechanisms . More spWe also observed larger P3 amplitudes elicited by painful than no-painful stimuli. It has been widely accepted that the P3, a positive shift of ERP wave that appears approximately 300 ms after the stimulus onset, reflects multiple cognitive processes, including top-down controlled attentional processes, cognitive evaluation of stimulus meaning, and the updating of representations in working memory . PascaliMore importantly, we found a linear relationship between the level of dispositional self-construal and amplitude difference of P3; the higher their level of the interdependent self-construal, the stronger their neural empathic responses to painful stimuli. Moreover, the level of interdependent self-construal could also positively predict behavioral ratings of perceived pain, but not self-unpleasantness. It has been suggested that empathy is an outcome of emotion identification or affect sharing processes, and these two processes are independent . In the In addition, some limitations of the present work should be noted. First, all participants who attended the present study are East Asians, whose interdependency is dominant . Thus, iTaken together, in accordance with previous studies demonstrating the modulation effect of temporary shifts in self-construal on the neural empathic responses to others’ pain , the preThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Hunan Normal University. The patients/participants provided their written informed consent to participate in this study.JC: study design, data analysis, and manuscript writing. BC: data analysis and manuscript writing. WL and YS: manuscript writing. LL, HS, and RW: data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway  requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.Because  Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme  predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum.A longstanding conundrum in  Treponema pallidum subsp. pallidum. Despite decades of intensive efforts toward its epidemiological control, syphilis continues to play a prominent role worldwide  the inability to cultivate the organism continuously in vitro   thl cells) , but it T. pallidum, we have focused on discerning salient structure-function relationships for T. pallidum’s membrane lipoproteins (LPs)   , 17–31. (LPs) 17–, 33, andore LPs) , 35. Our LPs) 17–. These elidum 21––28, 30, m 21––27–, 36–39. T. pallidum encounters numerous essential host-derived nutrients and exploits many of its periplasmic membrane LPs as components of transport systems  and cytoplasmic  cellular compartments. This includes a cytoplasmic flavin salvage pathway (Rhodobacter nitrogen fixation (RNF)-type energy conservation system T. pallidT. pallidum fulfills its energy requirements solely from the glycolytic processing of glucose    that wouradient)  7, 21, radient) . Finallyogenesis . In the ogenesis  Fig. 1)T. pallid, radientT. pallidum has been predicted by our previous in silico analysis  (tp0037 is annotated as a d-lactate dehydrogenase (d-LDH)  exploited by the pathogenic spirochete as it disseminates under low oxygen tension and encounters changes in external nutrient sources as part of its stealth pathogenicity. The possible origin of d-lactate as a substrate for the acetogenic energy conservation pathway in T. pallidum is discussed.The existence of a flavin-based energy conservation pathway in analysis , 21. Howogenesis , then thofactor) , 43. In  (d-LDH) . We crysWe hyperexpressed full-length TP0037 and purified it to apparent homogeneity. The protein yielded crystals that diffracted well, and molecular-replacement phasing followed by standard refinement approaches resulted in a model for the protein at a resolution at 1.38 Å. All validation and data quality statistics indicated that the data and the model were of high quality . The difThe TP0037 crystal structure featured two domains with a cleft between them , we term10.1128/mBio.02249-20.1FIG S110-helices are shown as green rectangles. Helices are associated with the two β-sheets shown, one in the NB domain, one in the C domain, and thus light green helices are below the plane of the respective sheet, while dark green ones are above the plane of the sheet. The labeling was done according to conventions for 2-hydroxyacid dehydrogenases   because of its role in binding the nucleotide-based cofactor NADor NADH) , harboreor NADH) , and theor NADH) .B-factors for their respective Cα atoms do not range much above the average B-factor for all atoms in the protein.The two domains were connected by a small region comprising the two points at which the main chain crossed from one domain to the other. They are defined by residues 300 and 301 and residues 101 and 102 . Because2 of buried surface area, and the dissociation of the dimer was calculated to have a free dissociation energy (ΔGdiss) of +36.1 kcal/mol, suggesting that the dimer is stable in solution.Two of these bilobed monomers were found in the asymmetric unit of the TP0037 crystals . The intd-lactate dehydrogenase from a Sporolactobacillus species   or a heuber 4XKJ ). The roand 1J49 , 50, 51;in vitro . SSM rand-lactate dehydrogenase from LbD-LDH was determined with NADH bound  . In LbD-LDH, NADH bound to the portion of the NB domain that faced the interdomain cleft. In essentially all cases, the superposition showed that LbD-LDH residues in contact with the NADH were matched by structurally homologous side chains from TP0037 having either identical or similar chemical natures  . We supe natures . It ther+ did not yield crystals suitable for structural characterization.A notable exception to the chemical correspondence between the NADH-binding residues of LbD-LDH and the putative ones of TP0037 occurred at contacts between LbD-LDH and the adenine moiety of NADH . LbD-LDHd-lactate dehydrogenase (LbD-LDH).Superpositioning also afforded us the opportunity to scrutinize the disposition of the residues responsible for catalysis in LbD-LDH and their structural homologs in TP0037. In LbD-LDH, these residues were E264, H296, and R235. The side chain carboxylate group of E264 was in contact with the imidazolium moiety of H296, with the guanidinium group of R235 poised nearby . E264, Hd-Lactate dehydrogenases are thought to exist as homodimers in solution, with no indication of ligand-induced oligomerization. However, this supposition has been inferred mostly from crystal structures  methodology (c(s) distributions were dominated by a single peak at 4.3 Svedberg units (S)  . No propnits (S) . The ave+ raised the melting temperature of TP0037 by approximately 4°C . Incubating 20 mM d-lactate (without NAD+) with the protein raised the melting temperature (mT) more modestly (ca. 1°C) but still significantly . l-lactate raised the temperature less (ca. 0.2°C) but measurably . These results confirm the binding of the necessary cofactor for catalysis and provide evidence for a strong preference to act on d-lactate rather than l-lactate.We also assessed ligand binding to TP0037 using differential scanning fluorimetry (DSF)  58). We. We58). mT values, i.e., 5 mM NAD+ raised the mT the most, with d-lactate showing a higher increase than l-lactate. We also observed that two of the mutants, Y101A and R235A, resulted in significant rises in mT with no additives . The origins of these substantial gains in stability are unknown but clearly indicate that these residues contribute to the metastability of the protein.Mutations of the putative active site residues Y101, R235, and H296 to alanine were made in anticipation of testing their respective activities (see the next section). We noted the same overall pattern on the respective d-lactate dehydrogenase activity, we performed an in vitro enzyme assay using the purified, monodisperse protein . For TP0037, we observed a specific activity in this assay of over 5,000 units/mg/ml. Using the same assay, only about one-thousandth of the activity was observed when using l-lactate as the substrate.To test whether TP0037 had  protein . The assAs a test to confirm that the putative active site residues Y101, R235, and H296 all had roles in catalysis, we individually mutated them to alanine and assessed the respective activities of the mutant proteins . Only Y1d-lactate dehydrogenases are widespread in bacteria, a large number of matches from other organisms were uncovered; we examined only the top one hundred, which were ostensibly the most closely related to TP0037. From these, we constructed a molecular phylogeny  were not included in this analysis because the amino acid sequences of their versions of TP0037 were identical to that of T. pallidum TP0037. The tp0037 gene appears to be co-operonic with one other gene, tp0038. By sequence homology, the product of the latter gene has been annotated as the enzyme IIC (EIIC) in a carbohydrate phosphotransferase system. EIICs are transmembrane proteins that transport carbohydrates across the bacterial cytoplasmic membrane and facilitate the transfer of a phosphate to them  for the generation of its ATP  that encodes at least one d-glucose ABC-type transporter (MglB-2)  , 60 and (MglB-2) , 34, 40.rylation .T. pallidum is its highly motile nature , motility, and tissue invasion -type redox pump (+), and the negative free-energy change  in this reaction drives the formation of a transmembrane ion gradient, which can then be used by the ATP synthase module to generate ATP . The gradient generated is requisite for driving additional ATP synthesis , followed by an oxidative decarboxylation of pyruvate to acetyl-CoA (and CO2) via a unique flavodoxin (Fld)-dependent pyruvate:flavodoxin oxidoreductase (PFOR) coupled with Fld reduction . As depicted in One notable difference between classical RNF redox pumps and that proposed for transfer , 72, 73.transfer . The ansT. pallidum’s Fld-PFOR is poised strategically at the interface of its d-lactate acetogenesis and the flavin-based RNF module . As denoted in + (−320 mV)   via the −320 mV) . In addi−320 mV) , therebyd-lactate to acetate in T. pallidum is likely to be the NAD+-dependent oxidation of the metabolite by d-lactate dehydrogenase (d-LDH) to form pyruvate and NADH , and the current study provides strong support for the notion that this enzyme can catalyze the starting point of d-lactate acetogenesis in T. pallidum. Although lactate is a common substrate for major groups of strictly anaerobic bacteria , where glucose supplies could be more limited. This metabolic flexibility may play a strategic role in T. pallidum’s stealth pathogenicity.The first step in the conversion of and NADH . The genand NADH , Table 2and NADH , and funand NADH  homologipyruvate . We therbacteria , 64, itsd-lactate for this pathway remains uncertain. However, bacteria in the human microbiome secrete this metabolite into the gastrointestinal tract  can be reasonably postulated in most human  tissues, including the gut epithelium, skeletal muscle, liver, kidney, neuronal tissues (including the brain), retina, and various blood cells , raising the possibility that this transporter is responsible for the import of d-lactate into T. pallidum  and Borrelia burgdorferi (Lyme disease) do not possess a TP0037 homolog; instead, they appear to metabolize l-lactate, as they both have an l-lactate dehydrogenase . This difference may partly limit the potential use of T. denticola and B. burgdorferi as heterologous systems for the study of T. pallidum genetics and biology.Despite the apparent broader implications of these findings across many bacterial species, d-lactate acetogenic energy conservation pathway is a key feature at the core of T. pallidum’s flavin-centric metabolic lifestyle, in which d-lactate is implicated as a previously unrecognized energy source. This addresses, at least in part, the decades-long energetics enigma imposed by the microaerophilicity of T. pallidum, which does not possess cytochromes, quinones, or ferredoxin as electron carriers. Therefore, the role of flavin as a central electron carrier in the d-lactate acetogenic energy conservation pathway , phosphate acetyltransferase (Pta), and acetate kinase (AckA). The genes putatively encoding PFOR, Pta, and AckA are tp0939, tp0094, and tp0476, respectively. Future structural, biophysical, and biochemical experiments on the products of these genes are warranted to confirm these respective functions. Our current studies also have laid a foundation for potentially exploiting the recently described long-term in vitro culture system for T. pallidum  competent cells were transformed with the ligated DNA products and selected for ampicillin resistance on LB agar plates. Cloning junctions/fragments were verified by DNA sequencing. E. coli BL21(DE3) cells were grown at 37°C in LB medium containing 100 μg/ml of ampicillin until the cell density reached an A600 of ∼0.6. The cells were then induced for ∼20 h with 0.6 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C and harvested, and the cell pellets were stored at −80°C. The procedures for expression and purification of the recombinant proteins were essentially as previously described (n-octyl-β-d-glucopyranoside (BOG).To produce a recombinant derivative of TP0037 in escribed , 29, 37.tp0037 sequence using the QuikChange site-directed mutagenesis kit (Agilent Technologies). The mutation was confirmed by DNA sequencing. The mutant protein was expressed and purified as described above. Protein concentrations were determined in storage buffer using UV absorption at 280 nm. Extinction coefficients were calculated from the protein sequences using the ProtParam tool of ExPASy server (www.expasy.org).For the construction of structure-guided recombinant TP0037 (rTP0037) variants, the R235A, Y101A, and H296A mutations were individually introduced into the plasmid carrying the wild-type N,N-methylenebisacrylamide [BIS]-Tris propane [pH 7.5], 20% [wt/vol] polyethylene glycol 3350 [PEG 3350]) after 7 days. The crystals were transferred to the stabilization buffer containing 200 mM sodium-potassium tartrate, 100 mM BIS-Tris propane (pH 7.5), 100 mM NaCl, 2 mM BOG, 20% PEG 3350, and various concentrations of ethylene glycol. For cryoprotection, crystals were serially transferred to solutions containing 5%, 10%, and 15% (vol/vol) ethylene glycol. After about 1 min in this final solution, the crystals were flash-cooled in liquid nitrogen and stored until data collection.Using the sitting-drop vapor-diffusion technique in 96-well plates with Structure 1 and 2 (Molecular Dimensions), JCSG plus (Qiagen), PACT (Molecular Dimensions), and Index (Hampton Research) crystallization suites and a crystallization robot , the initial crystallization conditions were determined at 20°C. Crystallization screens for TP0037 with a protein concentration of ∼20 mg/ml (in buffer A) yielded diffraction-quality crystals in C7 well of Structure 1 and 2 suite -2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans  wasdn/dc value of 0.1887 ml/g. The DLS data were analyzed using Dynamics, which was used in the regularization mode to calculate hydrodynamic radius distributions as shown in All DLS and SLS experiments were carried out in a Dynapro Nanostar instrument (Wyatt Technologies) at 25°C in storage buffer. The sample (5 μl at 1.54 mg/ml) was dispensed into a quartz cuvette, and the scattered light intensity and fluctuations thereof were simultaneously monitored. The results reported were from three technical replicates on the same sample. SLS data were analyzed in Dynamics version 7.5.0.17 (Wyatt Technologies) using a c(s) model available in SEDFIT  studies were conducted in a Beckman-Coulter XL-I ultracentrifuge at 20°C. Volumes of 400 μl of various concentrations of TP0037 in storage buffer were loaded into the sample sectors of charcoal-filled Epon centerpieces that were positioned between sapphire windows in standard aluminum housings. The same volume of storage buffer without protein was applied to the sample sectors. The assembled and sealed AUC cells were inserted into an An50-Ti rotor, which was then placed into the centrifuge. The samples were allowed to equilibrate at the experimental temperature for 2.5 h prior to centrifugation. The experiment was commenced by accelerating the rotor to 50,000 rpm; after the target rotor speed had been achieved, data acquisition was initiated utilizing the absorbance optics tuned to 280 nm. Data were collected overnight. The data were analyzed using the n SEDFIT , 57, witd-lactate or l-lactate. Samples were heat denatured from 4°C to 85°C in 0.5°C steps. The protein unfolding curves were monitored from the differential fluorescence changes (−ΔF) of protein-bound SYPRO Orange. The first derivative values (−ΔF/ΔT) reported by the instrument’s software were used to find the minimum value. The derivative data from this temperature, along with the preceding and succeeding five data points were fitted with a second-degree polynomial, and this fitting equation was analyzed to find the true minimum, i.e., the point at which the first derivative of the polynomial equation was 0, which was taken as the mT. All such experiments were performed in triplicate, and the reported mT values were the means of the replicates.To assess protein stability in the presence of ligands, differential scanning fluorimetry (DSF) of TP0037 and its variants were performed in a 96-well PCR plate (Bio-Rad) using a real-time PCR instrument (Bio-Rad). For binding assays, ligands were prepared freshly in sterile water. Each 25-μl standard assay mixture in a 96-well PCR plate contained 20 μl purified TP0037 and SYPRO Orange  at 5× concentration in a storage buffer. Five microliters of ligands were added in each well to final concentrations of 5 mM NAD and 20 mM either d-lactate dehydrogenase assay kit and Amplite colorimetric l-lactate dehydrogenase assay kit; AAT Bioquest) according to the manufacturer’s instructions. Briefly, for the d-lactate assays, the provided NAD was suspended in 100 μl to form a 200× solution. All reactions were carried out in the wells of 96-well plates; each well contained 100 μl, composed of 0.5 μl NAD, a volume needed to supply the desired protein mass , with the remainder of the volume comprising the manufacturer’s supplied assay buffer. For the l-lactate assays, an identical procedure was followed, except that 1 μg of the subject enzyme was added. The assays were incubated at 37°C for 30 min, followed by detection by monitoring increase in the A575/A605 ratio in a SPECTRAmax Plus plate reader (Molecular Devices). This value was compared to a standard curve  to arrive at the number of units of activity, which we normalized by the final concentration of the protein in the assay.Lactate dehydrogenase activity was assayed using commercially available kits . Custom


Acute appendicitis is the most frequent disease requiring emergency surgery and has an estimated lifetime risk of 6.7 to 8.6%.3The COVID-19-pandemic has impacted many lives and affects healthcare systems globally. To provide sufficient capacity for SARS-CoV-2 patients, elective surgery cases have been postponed in Germany from March 16, 2020, to May 2020.An increasing number of perforated appendicitis cases since March 16, 2020, prompted the question whether this increase can be ascribed to delayed initial medical contact after the onset of symptoms as a result of fear of contracting COVID-19 or whether there was a patient shift in our catchment area.This is a retrospective, single-center study, approved by the local ethic committee with analysis of all patients treated for acute appendicitis in a 10-week period from March 16 to May 31 in 2018, 2019, and 2020 were considered for the study. Informed consent was obtained from all participants.Data are available in SPSS v. 26.0.0.1  and were presented as means ± SD.p = 0.039). Treatment-related characteristics are listed in Table In total, 143 patients  were operated for acute appendicitis. Table Our clinical impression of seeing more complicated events of different entities, especially perforated appendicitis, was proven by this analysis. No relation between SARS-CoV-2 infections and appendicitis has been described so far.p = 0.039).We found a statistically significant increase in perforations during the recent period (Lessons to be considered in the second wave: Especially in the actual situation of increasing number of SARS-CoV-2 infections surgeons and institutions need to be prepared for advanced intraoperative findings, more intensive monitoring, more complications, and longer hospital stay in patients with acute appendicitis.


Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three  were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases.Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by  Xanthomonas species: X. euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri . He. He10]. ensitive .In vitro experiments where X. euvesicatoria 91–106 (pLAFR119) and X. euvesicatoria 91–106 (pLAFR119-ORF5) transconjugants were co-inoculated in nutrient broth with 91–118ΔBcnBC to determine sensitivity to Bcn-A. X. euvesicatoria 91–106 (pLAFR119-ORF5) reached concentrations of 105 to 106 CFU/mL in the presence of Bcn-A, whereas bacterial population of X. euvesicatoria 91–106 (pLAFR119) co-inoculated with 91–118ΔBcnBC had more than a thousand-fold lower viable cells/ml after 9 h  was recovered at an average of 1.5 log units higher than X. euvesicatoria 91–106 (pLAFR119-ORF5) when each was co-inoculated with X. perforans 91–118ΔBcnBC in leaf tissue (fter 9 h . In addif tissue . These rX. euvesicatoria ME90 (pXV519) cells were concentrated with 50 kDa and 100 kDa centriprep centrifugal filter units, . Bactericidal activity was detected in supernatants containing proteins 50 kDa and higher (Supernatants of d higher .X. euvesicatoria ME90 (pXV519), cellular proteins, supernatant and a mix of cellular proteins and supernatant were prepared by sonicating cells. The sonicated cell fraction was filtered through a 0.22 μm filter to eliminate bacteria. Bacteriocin activity was checked by deferred antagonism assay. The cell fraction did not have activity while Bcn-A activity was found in the supernatant  was obtained. The transconjugant was tested for Bcn-A activity. Bcn-A activity was observed in the wild-type but eliminated when expressed in the type II secretion mutant (An n mutant .ORFA) was submitted for homology modeling and the best model is shown in H. sapiens teneurin 2 (PDB ID: 6FB3), H. sapiens teneurin-3 (PDB ID: 6FAY), Photorhabdus luminescens tripartite toxin (PDB ID: 4O9X) and the BC component of the ABC toxin from Yersinia entomophaga (PDB ID: 4IGL). The amino acid sequence identities of Bcn-A to each of these templates ranged from 10% (4IGL) to 22% (6FAY). The teneurin family of proteins are large (~2800 amino acids) membrane anchored proteins responsible for cell-cell adhesion and are thought to have originated from the horizontal gene transfer of a bacterial YD-repeat toxin to an early heterotrophic eukaryote [The full 1398 amino acid sequence of Bcn-A  is predicted to cap the YD-barrel and extend into the core of the barrel domain. The Bcn-A homology model suggests that the protein serves as a membrane protein recognition and binding protein, potentially interacting with other subunits as is the case in the ABC toxins. Based upon the model, we hypothesize that Bcn-A interacts with a protein in the outer membrane of KpnI/EcoRI insert was subcloned into pLAFR3 and designated pLB5.8  in the forward direction (bcnB was determined to consist of 1398 nucleotides (GenBank Accession AB302849) and is predicted to encode a protein of 466 aa (48.5 kDa). Bioinformatic analysis predicts that Bcn-B has non-cytoplasmic signal peptide with an extracellular localization. The predicted amino acid sequence of Bcn-B is more than 98% identical to proteins from several other Xanthomonas species such as X. axonopodis, X. phaseoli, X. alfalfa, and X. campestris.In order to identify the Bcn-B irection . Based ubcnC gene was performed in pLAFR119. Given that plasmid pXV5.1 actively expressed Bcn-C without aid of the pL lac promoter  gave very slight bacteriocin activity compared to under direction of the lac promoter (bcnC gene was determined to consist of 1089 nucleotides (GenBank Accession AB302850) and expected to encode a 362 amino acid (38.2 kDa) protein. Bioinformatic analysis of Bcn-C predicts a non-cytoplasmic signal peptide. The deduced amino acid sequence of Bcn-C shares >97% identity with predicted metalloprotease proteins from X. euvesicatoria, X. axonopodis pv. citrumelo, and X. citri.In order to identify the Bcn-C ORF, directional cloning of the promoter , the natpromoter . The bcnX. perforans ME90 expressing Bcn-A (ME90 (pXV519)) was negative; however, X. perforans ME90 expressing bcnB (ME90 (pXV442)) and bcnC (ME90 (pXV120)) produced clear zones , and Bcn-C, ME90 (pXV120), by measuring protease activity after treatment by the serine protease inhibitor PMSF. Bcn-B activity was inhibited following treatment with PMSF while Bcn-C activity was unaffected, indicating that Bcn-B is a serine protease  as expecNo homologues for the initial 213 amino acids of Bcn-B were identified via BLAST searches of any available database. Submission of the amino acid sequence of Bcn-B to the Phyre2 homology modeling server did not result in any models generated that contained the initial 228 amino acids; however, the RaptorX homology modeling server did generate models for the initial 213 amino acids, indicating that two domains were present in this region , one spaAchromobacter lyticus. In addition, both algorithms predicted a small S1 pocket  contains a domain homologous to serine proteases. Submission of the amino acid sequence of Bcn-B to homology modeling servers resulted in models that contained a serine protease domain in that region, with a catalytic triad consisting of Asp337, His289, and Ser414 . Both th1 pocket  for the Grifola fondosa reveals that the zinc coordinating residues are predicted to occupy identical spatial positions, with an RMSD of less than 1.044Å for the aligned 108 atom pairs between the proteins.The Phyre2 algorithm was unable to model the 44 amino acids at the amino terminus of Bcn-C, however both the Phyre2 and RaptorX algorithms were able to confidently model the remainder of the protein. As shown in X. perforans. For Bcn-A, a five gene locus was identified that contributed to production of and immunity from bacteriocin function. Disruption of ORFA, ORF2 and ORF4 abolished Bcn-A activity based on deferred antagonism assay suggesting Bcn-A is part of a multiple component family of bacteriocins. The protein has significant sequence identity at the amino acid level with an ABC toxin from Yersinia entomophaga. This information and the predicted localization of each ORF in the inner and/or outer membrane of the cell suggests that these ORFs make up the necessary parts of a three component system  of a typical Gram-negative bacteriocin [ORFA gene product), whereas ORF5 encodes the protein responsible for the immunity function. ORF3 appears to be involved in production of Bcn-A given that there was a mild reduction in antagonism associated with disruption of ORF3. Bcn-A was only detected in supernatants and not at detectable levels in the cell fraction of Bcn-A producing X. perforans cells, suggesting Bcn-A is activated upon secretion. Therefore, proteins encoded by ORF2, ORF3, and ORF4 appear to play secondary roles such as in transport, modification, or secretion of Bcn-A. An ORF1 was predicted upstream of the Bcn-A genes  [E. coli genome from another organism with high GC content. The largest ORF of these elements spans the core and extension regions and is approximately 4.1-kb in length. This ORF is directly followed by three smaller ORFs . This core, like the putative bcnA gene product, is a high molecular weight, hydrophilic, devoid of a signal sequence protein, and contains 28 copies of an almost identical motif to that found in the C-terminal region of Bcn-A and WapA. A number of secreted ligand binding proteins have been identified as having similar motifs, which are believed to be involved in carbohydrate binding. These include a number of toxins, where the motifs are involved in target recognition [Clostridium difficile, are involved in interactions with the oligosaccharide components of receptor molecules on target cells [Serratia marcescens, to sensitive cells was blocked in RfaQ mutants which are impaired in core LPS biosynthesis [ORFA gene product. The key elements of these motifs  are present in Bcn-A. This motif has been identified in a discrete C-terminal portion of Bcn-A, which may be suggestive of a domain organization for this protein. Several high molecular weight toxin molecules, including bacteriocins, are organized into discrete domains, in which each has a different function. The domains usually have distinct binding and catalytic functions.Rhs elements were originally identified in bination . These ae “core” , encodesPF05593) . They alognition . The C-tet cells . Severalet cells ; howeverE. coli attributed no known function to these elements, a parallel between these elements and the genetic determinants for bacteriocin production by E. coli was noted [E. coli strains used for routine culturing [Carnobacterium piscicola strain LV17 produces two bacteriocins, carnobacteriocins BM1 and B2. The gene for immunity to B2, was located downstream of the B2 structural gene, and conferred immunity only to this bacteriocin [wapA, are non-essential to the cells that produce them [E. coli strains, it was proposed that they may play a role in the natural ecology of the cell. Perhaps the finding that they are toxic provides evidence for this. Bcn-A determinants are also not universally distributed among Xanthomonas campestris pathovars and Bcn-A negative mutants are viable, indicating a nonessential role for this compound. However, Koskiniemi et al. [rhs+/wapA+ cells from autoinhibition. Interestingly orf5, the immunity gene, does not have homology to any known proteins. Previously bcnB was localized to a 5.9 kb fragment [Although initial reports on Rhs elements of as noted . Colicinulturing . A shortteriocin . Rhs eleuce them ; howeveri et al.  demonstrfragment . Only twfragment .Bcn-C was previously localized to a 1.7 kb fragment . Two posBcn-B and Bcn-C were tested for protease activity based on homology data. Our findings show that both Bcn-B and Bcn-C exhibited protease activity; however, Bcn-B produced smaller cleared zones than Bcn-C.